In 2009, a new family of adenylyltransferases, defined by the presence of a 'Fic' domain, was discovered to catalyze the addition of adenosine mono-phosphate (AMP) to RhoGTPases. This adenylylation event inactivates RhoGTPases by preventing them from binding to their downstream effectors. The goal of this proposal is to investigate the functional repertoire of the Fic (filamentation induced by cyclic AMP) family of enzymes and understand the role of adenylylation as a post-translational modification in microbial pathogenesis and cellular signaling. Fic domains contain an HPFxxGNGR motif and are conserved from bacteria to humans. In E. coli, the Fic protein controls bacterial cell division, as a mutation in the fic gene results in aberrant septation3. Additionally, bacteriophage P1 encodes a Fic protein called Doc that is involved in stress survival4. We reported that the Fic domain(s) of the protein IbpA from the pathogenic bacterium Histophilus somni uses ATP to adenylylate and inhibit mammalian RhoGTPases, RhoA, Rac1 and Cdc42. This event induces host cytoskeletal collapse, which allows H. somni to breach alveolar barriers and become septicemic. The adenylylation modification occurs on a tyrosine in the switch1 region of these GTPases, which is critical for function. We further demonstrated that the only human Fic domain-containing protein, HYPE/FicD, also has the ability to add AMP to RhoGTPases in vitro. Further, the secreted toxin VopS from Vibrio parahaemolyticus has been shown to catalyze the addition of AMP to a conserved switch1 threonine in the same family of GTPases. Another variation on the Fic domain was reported for the Legionella pneumophila type IV secreted protein AnkX, where the catalytic His of the Fic motif was required for breakdown of the golgi network. While it remains to be determined if AnkX, Fic and Doc also function as adenylyltransferases, these findings suggest that Fic-mediated adenylylation represents a new signaling paradigm that functions to alter the activity of the modified substrate and mediate protein-protein interactions. Over 3400 Fic domain-containing proteins are known, and our understanding of this large enzymatic family comes from the study of just two bacterial proteins, IbpA and VopS. Using innovative approaches, including the use of AMP-specific antibodies and ATP- analogs to capture adenylylation events, this study will take a 3-pronged approach to understanding the role of Fic-mediated adenylylation in regulating prokaryotic and eukaryotic signaling events. Specifically, we will 1) Determine the functional conservation of the Fic motif; 2) Elucidate the role of E. coli Fic in bacteria; and 3) Elucidate the role of HYPE in eukaryotic signaling. This study will establish parameters to define a Fic protein as a functional adenylyltransferase, and determine whether Fic proteins from evolutionarily distinct organisms use adenylylation as a conserved mechanism for regulating signal transduction networks.